Diacetyl by Wine Making Lactic Acid Bacteria

Agric. Biol. Chem., 49 (7), 2147-2157, 1985 2147

Transformation of Citric Acid to Acetic Acid, Acetoin and Diacetyl by Wine Making Lactic Acid Bacteria Yoshimi Shimazu, Mikio Uehara and Masazumi Watanabe Food Research Laboratory, KikkomanCorporation, 399 Noda, Noda-shi, Chiba 278, Japan Received January 18, 1985

Adecrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC27307, a malo-lactic bacterium, grownon the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malo- lactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid. Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cell- free extract were optimal at pH 6.0. Divalent cations such as Mn2+ , Mg2+ , Co2 + and Zn2 + activated the citrate lyase. The citrate lyase was completely inhibited by EDTA,Hg2+ and Ag2 +. The acetoin formation from pyruvic acid was significantly stimulated by thiamine pyrophosphate and CoCl2, and inhibited by oxaloacetic acid. Specific activities of the citrate lyase and acetoin formation were considerably variable amongthe six strains of malo-lactic bacteria examined. Someactivities of irreversible reduction of diacetyl to acetoin were foundin the cell-free extracts of four of the malo- lactic bacteria strains and the optimal pH was 6.0 for this activity of Leu. mesenteroides.

Malo-lactic fermentation (MLF) in wine lished on the correlation between the levels of making is the process of conversion of malic acetoin and diacetyl in malo-lactic fermented acid to lactic acid and carbon dioxide by the wines and their sensory evaluation.16 ~20) action of certain lactic acid bacteria.1~4) This paper describes the cleavage of citric Furthermore, MLFcontributes not only to the acid to oxaloacetic and acetic acids, and the reduction of high acidity of wines, but also to subsequent formation of acetoin, diacetyl and the enhancement and complexity of wine acetic acid from pyruvic acid by intact cells flavors.5 ~8) or cell-free extracts of malo-lactic bacteria iso- In previous papers9'10) we reported that lated from fermenting wine musts. citric acid in addition to malic acid in must was considerably decomposedwith the occurrence of MLFand the amounts of acetoin in malo- MATERIALS AND METHODS lactic fermented wines increased markedly. Microorganisms. Six bacterial strains isolated from The mechanism of the formation of acetoin malo-lactic fermented musts were used. Leuconostoc and diacetyl by Streptococcus and Leuconostoc mesenteroides subsp. lactosum ATCC27307 (Nonomura 234),21) Leu. infrequens ATCC 27308 (Nonomura 83)21) species in cheese, butter and milk products has and Leu. dextranicum subsp. vinarium 721) were kindly been studied in detail by several workers.ll ~15) donated by Prof. H. Nonomura (Department of On the other hand, no report has yet been Fermentation Technology, Yamanashi University). Leu published, on the mechanismof the transfor- oenos PSU-122) was obtained from Tri Bio Laboratories, mation of citric acid to acetoin and diacetyl in Inc. (U.S.A.). Lactobacillus hilgardii BC2was a generous MLF, although many reports have been pub- gift from the Institut D'oenologie, Universite de 2148 Y. Shimazu, M. Uehara and M. Watanabe

Bordeaux, France. Lac. brevis B-9 was isolated from malo- 30°C for 30min, the reaction was stopped by heating in a lactic fermented must at our laboratory and classified by boiling water bath for 5min. The amount of acetoin was us. Leu. mesenteroides subsp. lactosum ATCC27307 was determined by gas chromatography. Oneunit of activity of used throughout to elucidate the transformation me- acetoin formation was denned as the amount of the cell- chanismfor citric acid. free extract required to liberate one /imol of acetoin from Saccharomyces cerevisiae IAM4274, from the Institute the substrate per minute at 30°C under the above of Applied Microbiology, Tokyo University, was used as a conditions. vinification starter. Determination of diacetyl reduction and acetoin dehy- Culture media and cultivation. The modified drogenation. Diacetyl-acetoin reduction was assayed by Nonomura's basal mediumfor lactic acid bacteria con- determining acetoin formed in a reaction mixture consist- tained 20g peptone, 5g yeast extract, 0.2g liver extract, ing of 1 mM diacetyl and 0.14mM NADHin 3.2ml of0.2m 0.2g Tween 80, 0.08g MnCl2, 0.2g MgSO4, 0.5g phosphate buffer (pH 6.0) and 0.3ml of the cell-free KH2PO4, 10g glucose and 5g fructose per liter of de- extract in a total volume of 3.5 ml. Acetoin-diacetyl dehy- ionized water. Ten grams of sodium citrate or sodium l- drogenation was assayed by determining diacetyl formed malate was added to the basal mediumfor pre-adaptation in a reaction mixture (final volume, 3.5ml) containing of the bacteria tested to each organic acid. Citrate and l- 1mMacetoin and 1mMNAD.Both mixtures were in- malate media were respectively adjusted to pH 5.0 and 6.5 cubated at 30°C for 30min. One unit of activity ofdiacetyl with lN-NaOH and autoclaved at 120°C for 20min. reduction or acetoin dehydrogenation was, respectively, Cultivation was carried out at 30°C for 3 days. defined as the amount of the cell-free extract required to liberate one /rniol ofacetoin or diacetyl from the respective Preparation of intact cells and cell-free extracts of lactic substrate per minuteunder the aboveconditions. acid bacteria. The cells of lactic acid bacteria were harvested from the culture broth by centrifugation at 12,000 x g Vinification. Grape must was prepared from Muscat for 30min at 4°C and then washed twice with 0.02m Bailey A grapes (36kg) harvested in 1982 in Yamanashi, potassium phosphate buffer (pH 6.0). The washed cells Japan. The must was treated with 50mgof sulfur dioxide were suspended in a small amount of 0.02m phosphate per liter of must in the form of potassium metabisulfite buffer (pH 6.0). The cells were disrupted at 0°C for 15 min and ameliorated with sucrose to a sugar concentration of with a Branson Sonifier Cell Disruptor 200P. The debris 22%. The prepared must (22 liters) was divided into two was removed by centrifugation at 34,000xg at 4°C for portions (each ll liters) and then each portion was 20min. The supernatant was dialyzed overnight against fermented on the skins at 23~25°C for 10 days. The first the same buffer (pH 6.0) at 4°C and used as the cell-free portion of the must (must a) was subjected to conven- extract. tional vinification without artifical inoculation with cul- For examination of the effects of metal ions on the tures of malo-lactic bacteria during fermentation. Wash- enzyme activity, 5.32g of solid ammoniumsulfate was ed cells (1 x 106 cells/ml) of Leu. mesenteroides cultivated added to 10ml ofa cell-free extract with stirring at pH 6.0. on the malic plus citric acids-medium were inoculated The precipitate collected by centrifugation was dissolved into the second portion of must (must b) on the fifth in 0.02m potassium phosphate buffer (pH 6.0) and dia- day (alcohol content, 10.4 vol%) of fermentation. lyzed overnight against the same buffer at 4°C. Wines A and B respectively obtained from musts a and b were stored at approximately 18°C for 30 days, Assay of citrate lyase activity. Citrate lyase activity was racked, sterile-filtered and finally bottled. assayed by determining acetic acid formed in a reaction mixture containing 5min sodium citrate, 1 mMMgCl2, Analyses. Chemical components of the test wines were 0.2m phosphate buffer (pH 6.0) and 0.3 ml of the cell-free analyzed by the methods of Hennig and Jakob.23) 2,4- extract in a total volume of 3.5ml. The reaction was Dinitrophenylhydrazine (DNPH) derivatives of pyruvic carried out at 30°C for 30min and stopped by the addition and oxaloacetic acids, and acetoin and diacetyl were of0.2ml of6n HC1solution. One unit of citrate lyase was identified by the descending paper chromatographic defined as the amount of the enzymerequired to liberate method used in a previous study.24) one /zmol of acetic acid from the substrate per minute at Citric, acetic, lactic, malic, formic, tartaric, succinic and 30°Cunder the above conditions. galacturonic acids were determined with a carboxylic acid analyzer model S-14 (Tokyo Rikakikai Co., Ltd., Tokyo) Assay of activity of acetoin formation from pyruvic acid. according to the EDC [l -ethyl-3-(3-dimethylaminopropyl) Acetoin formation from pyruvic acid by a cell-free extract carbodiimide hydrochloride] method of Shimazu et al.25) was assayed as follows. To 3.2ml of 0.2m phosphate l- and D-Lactic, and pyruvic and oxaloacetic acids were buffer (pH 6.0) containing 10mMsodium pyruvate, 1 mM assayed enzymatically as described previously.24) Acetoin CoCl2 and 0.5mM thiamine pyrophosphate (TPP) was and diacetyl were determined using a JEOLgas chroma- added 0.3ml of the cell-free extract. After incubation at tograph model JGC-1100 (Japan Electron Optics Transformation of Citric Acid by Wine Making Lactic Acid Bacteria 2149 Laboratory Co., Ltd.).26) Protein in the cell-free extracts Table I. Compositions of Muscat Bailey A Wine was determined by the method of Lowry et al.21) with Inoculated with or without Leu. mesenteroides bovine serum albumin as a standard. subsp. lactosum ATCC27307

RESULTS AND DISCUSSION Components Wine A Wine B Citric acid (g/liter) 0.46 0. 19 Composition of test red wine inoculated with or Lactic acid (g/liter) 0.22 2.27 without malo-lactic bacteria Malic acid (g/liter) 3.32 0.07 Acetic acid (g/liter) 0. 15 0.57 The conversion ofcitric acid and the fate of Acetoin (mg/liter) 6.00 1 1.00 various components in the test Muscat Bailey Diacetyl (mg/liter) 0 1.09 4 wine with or without inoculation of malo- lactic bacteria were examined. The compo- Alcohol (vol%) 1 1.3 1 1.4 sition of wine B made from must b inoculated Specific gravity 0.996 0.995 Extract 3.02 2.8 1 with washedcells of Leu. mesenteroides at the pH 3.25 3.45 middle stage of the fermentation on the skins is Total acids (g/liter)fl 8.20 6.32 shown in Table I. The small amount (0.22g/ Tartaric acid (g/liter) 1.69 1.64 liter) of lactic acid found in wine A from must Succinic acid (g/liter) 1.55 1.35 a indicates that MLFdid not occur in this wine Galacturonic acid (g/liter) 1.95 1.65 Total nitrogen (mg/liter) 23 1 230 process. On the other hand, the occurrence of Tannin (mg/liter) 950 905 MLFin wine B was shown by the disap- Color 430nm (x 5> 0.213 0.167 pearance of malic acid and the significant Color 530nm (x 5) 0.307 0.204 production of lactic acid (2.27g/liter). More Musta subject to the conventional vinification method acetic acid, acetoin and diacetyl and less citric and must b inoculated with washed cells of the malo-lactic acid were found in wine B than in wine A. bacterium were fermented on the skins at 20°C for 10 days Most diacetyl accumulated would be due to and pressed, followed, by storing at 18°C for 3 months. the action of the bacterium inoculated. Fermented wine Afrom must a and wine B from must b were filtered through 0.45 /im membrane filters and then Furthermore, malo-lactic fermented wine B analyzed. contained a smaller amount of tannin and a Expressed as the amount of tartaric acid. showed a lower optical density (430nm, 530nm) than wine A for which malo-lactic fermentation did not occur. ined with intact cells of Leu. mesenteroides In conclusion, citric acid in wines was re- subsp. lactosum. Washed cells (70mg dry cell markably decomposedand acetic acid, acetoin weight) of the lactic acid bacterium grown on and diacetyl were subsequently formed by the citric-medium were suspended in 25 ml of MLF. 0.1m glycine-HCl buffer (pH 2 to 4) or 0.2m phosphate buffer (pH 5 to 8) containing 15m Effect of pHon transformation ofcitric acid by sodium citrate as the substrate and then in- intact cells cubated at 30°C for 60min. The metabolic The preceding results suggest that the products of the substrate in the incubated marked formation of acetoin and diacetyl in broth were determined (Fig. 1). Citric acid was malo-lactic fermented wine making may be almost completely decomposed between pH attributed to the dissimilation of citric acid 2.0 to 6.0 and acetic acid was formed in by malo-lactic bacteria. More recently, parallel with the decomposition ofcitric acid in Lonvaudfunel et ai28) reported that large the whole pH range. These findings suggest amounts of d(-)lactate and acetate, and that the uptake of citric acid into the cells is smaller amounts of acetoin plus diacetyl were controlled by a pH-dependent system of the produced in the citrate-medium by malo-lactic cell membrane.29) bacteria. Therefore, the effect of pH on the d(-) and L(+)Lactic acids in the reaction transformation of citric acid was first exam- mixtures were respectively determined by en- 2150 Y. Shimazu, M. Uehara and M. Watanabe

zymatic methods.24* Since formation of and almost constant, most lactic acid was the L(+)lactic acid was very low (0.1 to 0.2mM) d(-)isomer. The above results revealed that malo-lactic bacteria can remarkably utilize citric acid and accumulate large amounts of 20 acetic acid, and small amounts of acetoin and 1 diacetyl in the normal pH range (3.0 to 4.0) of - 15 'o_ -o ° "°-~-- - 6 table wines. i10 y^sK '4I Transformation of citric, pyruvic, oxaloacetic and L-malic acids by intact cells /^^ ' 1'3 Washedcells of Leu. mesenteroides in 25 ml 0 -5- 1 . . . . u. o of 0.2 m phosphate buffer (pH 5.0) containing 25 one of these acids (each 15 him) were incubated - 12 at 30°C for 60min and the changes in the four i 20 ^^^-^^^ -10 i substrates were examined. The transformed 1 ^^^^\ - 8 "S product from citric acid was mostly acetic

a X#^ - 6f acid, with some acetoin, a little diacetyl and D(-)lactic acid, and traces of L(+)lactic, oxa- o 10 > \NX 2 I \\å à"1 loacetic and pyruvic acids (Table II). Acetoin and diacetyl were also main products from y-z1 citric, pyruvic or oxaloacetic acid. Less acetoin 0L , , 1 , , LJ0 and diacetyl were found and more pyruvic acid 2 3 4 5 6 7 8 (3.56mM) remained in the reaction mixture PH containing pyruvic acid as the substrate. On Fig. 1. Effect of pH on the Formation of Acetic and Lactic Acids and Acetoin plus Diacetyl from Citric Acid the other hand, pyruvic acid formed from by Intact Cells of Leu. mesenteroides subsp. lactosum citric or oxaloacetic acid as the substrate was ATCC 27307. little (0.04 to 0.05mM) and more acetoin and Washed cells (70 mg dry cell weight) of Leu. mesenteroides diacetyl were accumulated in the broth. From subsp. lactosum ATCC27307 were suspended in 25ml of the above results it is possible to assume that 0.1 m glycine-HCl buffer (pH 2 to 4) and 0.2m phosphate the formation of acetoin and diacetyl is due to buffer (pH 5 to 8) containing 15mMsodium citrate at the the transformation of pyruvic acid. Although indicate pHs, and the reaction mixtures were incubated at l(+) and d(-)lactic acids were remarkably 30°C for 60min. #-#, citric acid; A-A, acetoin plus diacetyl; O-O, produced by so-called MLF, acetoin and di- acetic acid; å¡-å¡,d(-) plus l(+) lactic acids. acetyl were not formed from L-malic acid.

Tableby IntactII. TransformationCells of Leu.of mesenteroidesCitric, Pyruvic,subsp.Oxaloaceticlactosum andATCCl-Malic27307 Acids

SubstrateutilizedAmounts

Acetic Products after incubation (itim) (him) d(-)Lactic acid L(+)Lactic acid acid Oxaloacetic acid Pyruvic acid Acetoin Citric acid (15 niM) 15.05 15.05 1.77 0.06 0.66 0.05 6.86 1.72 Pyruvic acid (15 him) 1 1.44 2.37 1.90 0.20 0.68 3.56 3.27 0.82 Oxaloacetic acid (15niM) 14.90 1.50 0.05 0.13 0.08 0.04 7.77 1.94 L-Malic acid (15mM) 14.90 0 2.63 10.51 0.10 0 0 0

Washedcells (70mg dry cell weight) ofLeu. mesenteroides subsp. lactosum ATCC27307 were suspended in 25 ml of 0.2m potassium phosphate buffer (pH 5.0) containing the above substrates (each 15 mM)and then the reaction mixtures were incubated at 30°C for 60 min. Metabolites after incubation were determined. Transformation of Citric Acid by Wine Making Lactic Acid Bacteria 2151 Namely, the above results indicate that the formation of acetoin and diacetyl by malo- "I /H lactic bacteria used in wine making is mainly 3. 5 / due to the dissimilation of not L-malic acid but 3.0 å / / citric acid. 2.5 å / / Transformation of citric acid by a cell-free extract s20' / / S The results in the preceding paragraphs led i'à"å å /// to the suggestion that the metabolism of citric acid by lactic acid bacteria used in wine mak- « 1.0 - * /// ^^^M ing may involve citrate lyase (EC 4.1.3.6)n ~14) which catalyzes the reversible aldol cleavage of citric acid to oxaloacetic and acetic acids as in the following equation [Eq. (l)].30) o JeE_-i 1 1 1 1- Citrate ^=^ Oxaloacetate+Acetate (1) 0 10 20 30 40 50 Incubation ( Min ) Therefore, the transformation of citric acid by Fig. 2. Effect of Concentration of the Dialyzed Cell-free the cell-free extract from Leu. mesenteroides Extract of Leu. mesenteroides subsp. lactosum ATCC was investigated to elucidate the mechanism of 27307 on Acetic Acid Formation from Citric Acid. metabolism of citric acid in the wine making Thereaction mixtures contained 5 mMsodiumcitrate and process. 1 mMMnCl2 in 3.2ml of 0.2m phosphate buffer (pH 6.0) and 0.3ml of the dialyzed cell-free extract in a total volume of 3.5ml. The concentrations of the cell-free Formation of acetic acid from citric acid by the extract were as follows. -A-, 3.0mg/ml; -#-, cell-free extract 6.0mg/ml; -O-, 9.0mg/ml; -å -, 12.0mg protein/ml. First, the effect of different concentrations After static incubation at 30°C for the indicated times, of the dialyzed cell-free extract on the for- acetic acid in the reaction mixture was determined. mation of acetic acid from citric acid was examined. The reaction was allowed to pro- ceed at 30°C after adding 0.3ml of the 100 å >A\ 90 / N^ respective cell-free extract (3.0, 6.0, 9.0 or 80 / \ 12.0mg protein/ml) to the reaction mixture 70 / \ (3.2ml) containing 5mMsodium citrate and 60 / \ 1mMMnCl2. Figure 2 shows typical time H 50 / \ courses of acetic acid formation from citric I 40" / \ acid by the cell-free extract at different protein | 30 / concentrations. The amounts of acetic acid I 20 - / \ 10 / formed increased in proportion to the increase o Li à" à" à" à" 1- in concentration of the cell-free extracts. 3 4 5 6 7 8 Acetic acid was formed almost linearly with PH the progress of the reaction. These facts in- Fig. 3. Effect of pH on Citrate Lyase Activity of the dicate that the formation of acetic acid from Cell-free Extract of Leu. mesenteroides subsp. lactosum citric acid is due to citrate lyase of malo-lactic ATCC 27307. bacteria. The reaction mixtures contained 5mM sodium citrate, l mMMnCl2 and 0.3ml of the dialyzed cell-free extract (6.0mg protein/ml) in a total volume of 3.5ml. The Effect of pHon citrate lyase activity reactions at the indicated pHs were statically carried out at Figure 3 shows the effect of pH on citrate 30°C for 30min. 0.1 m glycine-HCl buffer (pH 3 to 4) and lyase activity of the dialyzed cell-free extract 0.2 m phosphate buffer (pH 5 to 8) were used, respectively. 2152 Y. Shimazu, M. Uehara and M. Watanabe

Table III. Formation of Acetic, Oxaloacetic, Pyruvic and Lactic Acids, and ACETOIN FROM ClTRIC ACID BY THE CELL-FREE EXTRACT OF Leu. mesenteroides subsp. lactosum ATCC27307

~.. . , Product (him) Citricacid v ' Co factors utilized . , (him) . ,j Acetic Oxaloacetic., Pyruvic., Oxaloaceticplus. ., Lactic., Acetoin . acid acid acid pyruvic acids acid

l mM Mn2+ 7.05 6.76 6.20 0.70 6.90 0.07 0 (0.96) (0.88) (0. 10) (0.98) (0.01) l mM Mn2+ 7.10 6.85 5.64 1.41 7.05 0.08 0.019 0.5mM TPP (0.96) (0.79) (0.20) (0.99) (0.01) (0.003) l mM Mn2+ 7.09 6.82 4.95 2.05 7.00 0.10 0.022 l mM Co2+ (0.96) (0.90) (0.29) (0.99) (0.01) (0.003) 0.5mMTPP

Figures in parentheses indicate molar ratios of products to citric acid utilized. To 3.2ml of 0.2 m phosphate buffer (pH 6.0) containing 50mMsodium citrate and metal ions and/or TPP at the indicated concentrations was added 0.3 ml of the cell-free extract (19.5 mgprotein/ml) from Leu. mesenteroides subsp. lactosum ATCC27307. The reaction mixtures were incubated statically at 30°C for 30 min, and then organic acids and acetoin were determined.

from the malo-lactic bacterium. Citrate lyase of the enzyme activity was respectively ex- activity was significantly low below pH 4.0 or pressed compared to the control level. 1 mM above pH 8.0 and optimal at pH 6.0, which iodoacetic acid, L-cysteine or 8-hydroxy- was appreciably lower (pH 7.4 to 7.6) than in quinoline showed no effect on the enzyme the case of Streptococcus diacetilactis reported activity. Sodium azide (1 mM)slightly activated by Harvey et al.14r) the enzyme. Effect of metal ions on citrate lyase activity Metabolites from citric acid The effect of metal ions on citrate lyase Metabolites from citric acid incubated with activity was also examined using the cell-free the dialyzed cell-free extract were determined extract salted out with ammoniumsulfate and to elucidate the transformation mechanismof dialyzed as described in Materials and citric acid ofmalo-lactic bacteria. Almost equi- Methods. Mn2+, Mg2+, Co2+ and Zn2+ were molar amounts of acetic and oxaloacetic acids, found to be efficient activators. Mn2+was the and traces of lactic acid and acetoin were most effective activator for the enzymeand up found as the metabolites of utilized citric acid to 1 mMthe enzymeactivity was dependent on (Table III). The stoichiometric data indicate the concentration of MnCl2.Onthe other that citric acid during wine making is split into hand, Ca2+, Fe2+, Fe3+ and Ba2+ were in- oxaloacetic and acetic acids by citrate lyase hibitory for the enzyme. The strongest in- (EC 4.1.3.6) of malo-lactic bacteria [Eq. (1)]. hibition by Ca2 + for the citrate lyase coincided The notable appearance of pyruvic acid in the with the result reported for Aerobactor reaction mixture supplemented with cocar- aerogenus.31 ) boxylase suggests the decarboxylation of oxaloacetic acid by this enzyme. Effect of inhibitiors on citrate lyase activity A significant amount of acetoin was pro- Either EDTAor mercury chloride at 1 mM duced from citric acid with the intact cells of completely inhibited the citrate lyase. In the the malo-lactic bacterium as described in the presence of 0.1mM silver nitrate, or 1mM previous paragraph. However, acetoin (ap- potassium ferrocyanide, 0-phenanthroline or proximately 0.02mM) was scarcely formed /?-chloromercuribenzoic acid, 5, 40, 81 or 94% from citric acid and a large amount of oxalo- Transformation of Citric Acid by Wine Making Lactic Acid Bacteria 2153

2.0 " f

(A) (B) ^ o i / ; i-5 - -

I1-°å / å // 1" [

oV^-vr^\0 3 6 9 , 12 ,' 0 Ir,15 30 45. 0 . 15 Jk,30 45 l-0 15 .3.0 ^-45

Concentration of cell-free extract Incubation ( Min ) ( mg protein / ml) Fig. 4. Formation ofAcetoin, Acetic Acid and Diacetyl from Pyruvic Acid by the Cell-free Extract of Leu. mesenteroides subsp. lactosum ATCC27307. In experiment (A), the reaction mixtures contained 10mMsodium pyruvate, 0.5 mMTPP and 1 niM CoCl2 in 3.2ml of 0.2m phosphate buffer (pH 6.0) and 0.3ml of the dialyzed cell-free extract (3.0, 6.0, 9.0 or 12.0mg protein/ml) in a total volume of 3.5ml. Incubation was statically carried out at 30°C for 30min. Symbols: (#), acetoin; (A), acetic acid; (å ), diacetyl. In experiment (B), the mixtures contained the above standard assay reagents in 3.2ml of 0.2m phosphate buffer (pH 6.0) and the cell-free extract (each 0.3 ml) in a total volume of 3.5 ml. After static incubation at 30°C for the indicated times, acetoin, acetic acid and diacetyl were determined. Symbols: Acetoin, (O) 12.0mg/ml; O) 9.0mg/ml; (#) 6.0mg/ml; (0) 3.0mg protein/ml. Acetic acid, (A) 12.0mg/ml; (A) 9.0mg/ml; (A) 6.0mg/ml; (A) 3.0mg protein/ml. Diacetyl, (å¡) 12.0mg/ml; (|J) 9.0mg/ml; (å ) 6.0mg/ml; (EB) 3.0mg protein/ml. acetic acid remained in the reaction fluid acid, transformation of pyruvic acid was also with the cell-free extract of this bacterium. examined. Therefore, it is possible to suppose that oxaloacetic acid, and elementary metabolite from Formation of acetoin, acetic acid and diacetyl citric acid with citrate lyase (EC 4.1.3.6), from pyruvic acid may suppress the subsequent acetoin forma- Acetoin, acetic acid and diacetyl were tion in the reaction with the cell-free extract. formed from pyruvic acid in proportion to the concentration of the cell-free extract. Figure 4 Formation of acetoin, diacetyl and acetic acid shows the respective time courses of the for- from pyruvic acid by the cell-free extract mation of acetoin, acetic acid and diacetyl Based on the above results, it can be con- from pyruvic acid. Acetoin and diacetyl were cluded that lactic acid bacteria used in wine produced linearly for 45min, but acetic acid making transform citric acid to acetic and formation became almost constant after oxaloacetic acids, the latter of which is then 15 min reaction. These results indicate that the decarboxylated to pyruvic acid. Therefore, in main products formed from pyruvic acid by order to elucidate the metabolic pathway of malo-lactic bacteria are acetoin, acetic acid acetoin and diacetyl formation from pyruvic and diacetyl. 2154 Y. Shimazu, M. Uehara and M. Watanabe

Table IV. Effect of Metal Ions on Acetoin preparation from the cell-free extract. Acetoin Formation from Pyruvic Acid by the Salting- formation without Co2 + was stimulated about out Cell-free Extract from Leu. mesenteroides five-fold compared to the control with over subsp. lactosum ATCC27307 0.5mM TPP. With 0.5mM TPP, acetoin for- Relative activity (%) mation from pyruvic acid was dependent on the CoCl2 concentration up to 1 mMand inde- None 100 pendent in the range of 1 to 3mM, but sup- Co2+ 161 pressed at over 3mMCoCl2. Ca2+ 117 Ba2+ 116 Effect of inhibitors on acetoin formation Mg2+ 111 Zn2 + 105 Acetoin formation from pyruvic acid with Mn2+ 102 the cell-free extract from Leu. mesenteroides Fe2+ 38 was inhibited 45% by 1 mML-cystein and 86 to Sn2+ 38 90% by silver nitrate (0.1 mM), iodoacetic acid Fe3 + 29 (1 mM), acetaldehyde (1 niM)34) and 8-hydroxy- Cu2 + 8 quinoline (l mM), but somewhat activated by The acetoin formation was determined by the standard 1 mMEDTA, />-chloromercuribenzoic acid, o- assay method with each metal ion at lmM. Relative phenanthroline, sodium azide and potassium activity is expressed as a percentage to the control (no ferrocyanide. metal ions). Metabolic pathway for pyruvic acid Effect of pHon acetoin formation Furthermore, some metabolic products The pH optimum was 6.0 for acetoin for- from pyruvic acid were examined in order to mation from pyruvic acid with the cell-free elucidate the mechanism of pyruvic acid con- extract of Leu. mesenteroides. Noacetoin was version. We confirmed that acetoin, acetic formed in this system below pH 5.0 or above acid, diacetyl and lactic acid were formed from pH 8.0. pyruvic acid under the optimal conditions as described in the preceding paragraph. The Effect of metal ions on acetoin formation molar ratio of the sum of acetoin, diacetyl, The effect of metal ions on acetoin for- acetic and lactic acids formed to pyruvic acid mation from pyruvic acid was further exam- utilized as the sole substrate was approxi- ined using a salting-out preparation from the mately 0.5 as shown in Table V. Therefore, it cell-free extract. Of the ten metal ions tested, was assumed that biosynthesis of acetoin from Co2+, Ca2+, Ba2+ and Mg2+ activated pyruvic acid by malo-lactic bacteria may be formation and Cu2+, Fe3+, Sn2+ and Fe2+ involved in the metabolic reaction in which a- inhibited acetoin formation in that order acetolactic acid is formed from two mol of (Table IV). Co2+ was the most effective pyruvic acid and decarboxylated.n'34'35) activator. Next, the effect of acetic or oxaloacetic acid was examined on the metabolism of pyruvic Effect of TPP and Co2+ on acetoinformation acid. No inhibition of pyruvic acid metabolism Singer et al.32) and Koike et al.33) have by acetic acid was found. However, 1mM reported that the decarboxylation of pyruvate oxaloacetic acid significantly suppressed the involved in the acetoin formation reaction formation of acetoin, diacetyl and acetic acid requires TPP. Furthermore, we found that from pyruvic acid by the cell-free extract of Co2 + was required for acetoin formation from Leu. mesenteroides. No acetoin or diacetyl was pyruvic acid. Therefore, the effects of the detected in the presence of 5mMoxaloacetic concentrations of TPP and CoCl2 on acetoin acid. These results support the preceding formation were examined using the salting-out speculation that acetoin formation from Transformation of Citric Acid by Wine Making Lactic Acid Bacteria 2155

Table V. Formation of Acetoin, Diacetyl and Acetic and Lactic Acids from Pyruvic OR OXALOACETIC ACID BY THE DlALYZED CELL-FREE EXTRACT OF Leu. mesenteroides subsp. lactosum ATCC27307

Pyruvic Products (him) Substrate Co factor utilized (him) Acetoin Diacetyl Acetic acid Lactic acid

Pyruvic acid (10 him) None 0.74 0. 10 0.04 0.23 0.02 Pyruvic acid (10him) TPP (0.5 mM) 2.73 0.59 0. 18 0.60 0.02 Pyruvic acid (10niM) TPP (0.5 mM) 3. 15 0.95 0. 17 0.42 0.15 CoCl2(1him) Pyruvic acid (10mM) TPP (0.5 mM) 3. 13 0.90 0. 16 0.41 0.05 CoCl2(1mM) NAD(1him)

Pyruvic acid (10him) TPP (0.5 mM) - 0.73 0. 1 1 0.30 0.05 Oxaloacetic acid (0.5 mM) CoCl2 (1 him) Pyruvic acid (10mM) TPP (0.5 mM) - 0.54 0.05 0.26 0.04 Oxaloacetic acid (1 mM) CoCl2 (1 mM) Pyruvic acid (10mM) TPP (0.5 mM) 2.99 0.92 0.16 1.43 0.03 Acetic acid (1 mM) CoCl2 (1 mM) Oxaloacetic acid (5 mM) TPP (0.5 mM) - 0 0 0.03 0.08 CoCl2(1mM)

The reaction mixtures consisted of pyruvic acid (10mil), oxaloacetic acid (0.5, 1.0 or 5mM)or acetic acid (l niM) solely or in combination as substrates in the absence or presence of TPP (0.5mM), CoCl2 (1 iiim) and NAD(1 him) in 3.2ml of 0.2m phosphate buffer (pH 6.0) and 0.3 ml of the cell-free extract (9.0mg protein/ml) of Leu. mesenteroides subsp. lactosum ATCC27307. Static incubation was carried out at 30°C for 30min, and then metabolic products in the incubated broths were determined.

Fig. 5. Proposed Pathway for Acetoin, Diacetyl and Acetic Acid Transformation from Citric Acid in Wine Making by Malo-lactic Bacteria. 2156 Y. Shimazu, M. Uehara and M. Watanabe

Table VI. Activities of Citrate Lyase and of acetic acid, acetoin and diacetyl in the Acetoin Formation of the Cell-free resultant wines. Extracts from Various Malo- lactic Bacteria Intertransformation of acetoin and diacetyl Specific activity The parallel formation of acetoin and di- Strain Citrate lyase acetyl from pyruvic acid was described in the (units/mgprotein) Lactobacillus hilgardii BC2 preceding paragraphs. Enzymic intertransfor- LeuconostocLactobacillus oenosbrevis PSU-1B-9 mation of acetoin and diacetyl was studied Leuconostocsubsp. vinariumdextranicum7 Leuconostocsubsp. lactosummesen teroidesATCC27307 using the dialyzed cell-free extracts of six ATCCLeuconostoc27308 infrequens 1.33 43.63 malo-lactic bacteria. Dehydrogenation of ace- 2.47 6.01 toin to diacetyl was not seen in any of the

29.90 strains. However, some activity of diacetyl

73.38 reduction to acetoin was detected in Lac. 6.99 17.37 1.70 brevis, Leu. oenos, Leu. dextranicum and Leu. 6.00 mesenteroides, and the activities were respec- 12.21 tively 0.59, 0.93, 1.01 and 0.53 (units/mg pro- 24.3 tein). The optimal pH for this reaction was ATCC: the American Type Culture Collection, found to be 6.0 with the Leu. mesenteroides Rockville, U.S.A. The specific activities of citrate lyase cell-free extract. Diacetyl reductase (EC and acetoin formation were assayed by the respective 1. 1. 1.5), which irreversibly converts diacetyl to standard assay methods. acetoin, has been found in butter and milk lactic acid bacteria, Streptococcus species36) citric acid may be inhibited by oxaloacetic and Lactobacillus casei.31'38) Diacetyl reduc- acid derived from citric acid with citrate lyase tase was also found in some strains of malo- (EC 4.1.3.6). lactic bacteria. The above results maysuggest Figure 5 shows the proposed pathway of that a part of acetoin accumulated in malo- citric acid metabolism by malo-lactic bacteria lactic fermented wines is formed by diacetyl used in wine making. Namely, citric acid is first reduction in addition to biosynthesis through split into acetic and oxaloacetic acids. The a-acetolactate from pyruvic acid. Diacetyl is latter acid is subsequently decarboxylated to recognized as an important aromacomponent pyruvic acid which is converted to acetoin, giving a butter-like flavor and as an off-flavor diacetyl and acetic acid. when over 3mg diacetyl per liter is present, especially in red wines.20) Based on the above Activities of citrate lyase and acetoin for- results, the selective application of malo-lactic mation amongvarious malo-lactic bacteria bacteria with higher diacetyl reduction activity Citrate lyase and acetoin formation from may enable reduction of the diacetyl contents pyruvic acid were compared amongsix typical of the resultant wines. strains of malo-lactic bacteria used in wine making. Both activities were considerably high Acknowledgments. The authors wish to thank in Leu. infrequens, Leu. mesenteroides subsp. Professor T. Tochikura of Kyoto University for his helpful lactosum and Lact. brevis (Table VI). These advice throughout this study. The authors also thank Dr. S. Nasuno for his help in preparation of the manuscript, results suggest that the extent of conversion of and Dr. F. Yoshida and Dr. D. Fukushima of Kikkoman citric acid in malo-lactic fermented wines may Corporation for their encouragement. be highly dependent on the malo-lactic bacteria that grow spontaneously during wine REFERENCES making. Through artificial induction of MLF, 1) M. Schiitz and F. Radler, Arch. MikrobioL, 91, 183 the use of malo-lactic bacterial strains selected (1973). properly mayenable us to control the amounts 2) M. Schiitz and F. Radler, Arch. MikrobioL, 96, 329 Transformation of Citric Acid by Wine Making Lactic Acid Bacteria 2157

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